Somatostatin Analogues

ABSTRACT

The invention provides cyclo[{4-(NH 2 —C 2 H 4 —NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Benzyl)-Phe], optionally in protected form, or a pharmaceutically acceptable salt or complex thereof, which has interesting pharmaceutical properties.

This application is a divisional of U.S. application Ser. No. 12/265,135filed Nov. 5, 2008 which is a divisional of application Ser. No.10/343,288 filed Aug. 26, 2003 now U.S. Pat. No. 7,473,761 which is aNational Stage of International Application PCT/EP01/08824 filed on Jul.30, 2001, which in its entirety is herein incorporated by reference.

The present invention relates to somatostatin peptidomimetics, a processfor their production and pharmaceutical preparations containing them.

More particularly the present invention provides the compound of formula

also calledcyclo[{4-(NH₂—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe], referredherein to as Compound A, as well as diastereoisomers and mixturesthereof, in free form, in salt or complex form or in protected form. Phgmeans —HN—CH(C₈H₅)—CO— and Bzl means benzyl.

Compound A in protected form corresponds to above molecule wherein atleast one of the amino groups is protected and which by deprotectionleads to Compound A, preferably physiologically removable. Suitableamino protecting groups are e.g. as disclosed in “Protective Groups inOrganic Synthesis”, T. W. Greene, J. Wiley & Sons NY (1981), 219-287,the contents of which being incorporated herein by reference. Example ofsuch an amino protecting group is acetyl.

When Compound A exists in complex form, it may conveniently be aCompound A bearing a chelating group on the side chain amino group ofPro and complexed with a detectable or radiotherapeutic element.Compound A bearing a chelating group is referred to hereinto asconjugated Compound A.

Examples of chelating groups include e.g. those derived frompoly-aminopolycarboxylic acids or anhydrides, e.g. those derived fromnon cyclic ligands e.g. diethylene triamine pentaacetic acid (DTPA),ethylene glycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid(EGTA), N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)and triethylenetetramine hexaacetic acid (TTHA), those derived fromsubstituted DTPA, e.g. p-isothiocyanato-benzyl-DTPA, those derived frommacrocyclic ligands, e.g.1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),or 1,4,7,10-tetraazacyclotridecane-N,N′, ,N″,N′″-tetra-acetic acid(TITRA).

The chelating group may be attached either directly or through a spacerto the side chain amino group of Pro_(—) Suitable spacers include thoseknown in the art, e.g. as disclosed in GB-A-2,225,579, for example thedivalent residue of an amino-carboxylic acid, for example β-Ala or adivalent residue derived from 6-amino-caproic acid.

Preferred chelating groups are those derived from DTPA, DOTA or TETA.Chelating groups derived from DTPA or DOTA are most preferred.

By detectable element is meant any element, preferably a metal ion whichexhibits a property detectable in vivo diagnostic techniques, e.g. ametal ion which emits a detectable radiation or a metal ion which iscapable of influencing NMR relaxation properties. By radiotherapeuticelement is meant any element which emits a radiation having a beneficialeffect on the conditions to be treated.

Suitable elements include for example heavy elements or rare earth ions,e.g. as used in CAT scanning (Computer axial tomography), paramagneticions, e.g. Gd³⁺, Fe³⁺, Mn²⁺ and Cr²⁺, fluorescent metal ions, e.g. Eu³⁺,and radionuclides, e.g. a radiolanthanide, particularly a γ-emittingradionuclide, β-emitting radionuclide, α-emitting radionuclide,Auger-e⁻-emitting radionuclide or a positron-emitting radionuclide e.g.⁶⁸Ga, ¹⁸F or 86Y.

Suitable γ-emitting radionuclides include those which are useful indiagnostic techniques. The γ-emitting radionuclides advantageously havea half-life of from 1 hour to 40 days, preferably from 5 hours to 4days, more preferably from 12 hours to 3 days. Examples areradioisotopes from Gallium, Indium, Technetium, Ytterbium, Rhenium,Terbium, Lutetium, Thallium and Samarium e.g. 67_(Ga,) 111In, ^(99m)Tc,¹⁶¹Tb, ¹⁶⁹Yb, ¹⁸⁶Re or ¹⁷⁷Lu.

Suitable β-emitting radionuclides include those which are useful inradiotherapeutic applications, for example ⁹Y, ⁶⁷Cu, ¹⁹⁹Re, 188Re,169Er, 121_(Sn,) ¹²⁷Te, ¹⁷⁷Lu, ¹⁴³Pr, ¹⁹⁸Au, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴²Pr or ¹⁵³_(Sm.)

Suitable α-emitting radionuclides are those which are used intherapeutic treatments, e.g. ²¹¹At, ²¹²Bi or ²⁰¹ _(Tl.)

Compound A may exist e.g. in free or salt form. Salts include acidaddition salts with e.g. inorganic acids, polymeric acids or organicacids, for example with hydrochloric acid, acetic acid, lactic acid,aspartic acid, benzoic acid, succinic acid or pamoic acid. Acid additionsalts may exist as mono- or divalent salts, e.g. depending whether 1 or2 acid equivalents are added to the Compound A in free base form.Preferred salts are the lactate, aspartate, benzoate, succinate andpamoate including mono- and di-salts, more preferably the aspartatedi-salt and the pamoate monosalt.

The conjugated Compound A may additionally exist in salt formsobtainable with the carboxylic acid groups when present in the chelatinggroup, e.g. alkali metal salts such as sodium or potassium, orsubstituted or unsubstituted ammonium salts.

The present invention also includes a process for the production ofCompound A. It may be produced in analogy to known methods, for example:

a) cyclising a linear peptide in protected, polymer-bound or unprotectedform in such a way that Compound A is obtained and then optionallyremoving the protecting group(s),b) to produce a conjugated Compound A linking together a chelating groupand the

Compound A in protected or unprotected form and then optionally removingthe protecting group, and recovering Compound A or a conjugated CompoundA thus obtained, in free form, in salt form or optionally complexed witha detectable or radiotherapeutic element.

It is generally not critical which amino acid is selected to be at theC-terminal position to start the peptide chain since the linear peptidewill be cyclized, provided only that the sequence of amino acids in thelinear peptide corresponds to that in Compound A. However there may beother factors which may prefer one starting amino acid over another.When Compound A is prepared by solid phase synthesis, the firstamino-acid is preferably attached to the resin, e.g. a commerciallyavailable polystyrene-based resin, through a suitable linker, e.g. alinker which is cleavable under mild conditions to keep the side chainprotection intact, e.g. SASRIN or an optionally substituted trityl basedlinker, for example 4-(hydroxy-diphenyl-methyl)-benzoic acid wherein onethe phenyl groups may optionally be substituted e.g. by Cl. The buildingup of the desired peptide chain may be effected in conventionnal manner,e.g. using amino-acid units wherein the terminal amino group isFmoc-protected, the side chain amino groups where present beingprotected with a different amino protecting group, e.g. Boc or CBO.Preferably the linear peptide is cyclized in such a way to produce abond between Tyr(4-Bzl)—OH and Phe, e.g.Phe-{4-(NHR₁—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp(R₂)-Lys(ε-NHR₃)-Tyr(4-Bzl)—OHor a functional derivative thereof, wherein each of R₁, R₂ and R₃ is anamino protecting group. The cyclisation step a) may conveniently beperformed according to known method, e.g. via an azide, an active ester,a mixed anhydride or a carbodiimide. Thereafter the protecting groupsare removed, e.g. by cleavage e.g. with trifluoroacetic or byhydrogenation.

The cyclisation of the peptide may also be performed directly on thesolid support, the first amino acid being in a Nα- and C-terminalprotected form and attached through a side chain, e.g. ε-amino functionof Lys or by backbone anchoring. The linear sequence is then synthesizedfollowing standard solid phase synthesis (SPPS) procedures. Aftercleavage of the C-terminal protection the peptide is cyclized e.g. asdescribed above. Thereafter the cyclic peptide is cleaved from the resinand deprotected.

If desired, the lateral chain present on Pro may be introduced on theamino acid prior to or after the peptide cyclisation step a). Thus, Proas a starting amino-acid or a starting linear or cyclic peptide whereinin each case Pro is ring-substituted by OH, may be converted to provideCompound A or the desired Pro unit or the corresponding linear peptide,respectively, wherein Pro is substituted by NHR₁—C₂H₄—NH—CO—O—.

The complexation of a conjugated Compound A may be performed by reactingthe conjugated Compound A with a corresponding detectable orradiotherapeutic element yielding compound, e.g. a metal salt,preferably a water-soluble salt. The reaction may be carried out byanalogy with known methods, e.g. as disclosed in Perrin, Organic Ligand,Chemical Data Series 22. NY Pergamon Press (1982); in Krejcarit andTucker, Biophys. Biochem. Res. Corn. 77: 581 (1977) and in Wagner andWelch, J. Nucl. Med. 20: 428 (1979).

The following examples are illustrative of the invention. Alltemperatures are in ° C.

Abbreviations:

-   AcOH=acetic acid-   Boc=tert.-butoxy-carbonyl-   Bzl=benzyl-   CBO=carbobenzoxy-   DIPCl=N,N′-diisopropylcarbodiimide-   DIPEA=diisopropylethylamine-   DMF=dimethylformamide-   DPPA=diphenyiphosphorylazide-   Fmoc=fluorenylmethoxycarbonyl-   HOBT=1-hydroxybenzotriazole-   Osu=N-hydroxysuccinimide-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran

EXMAPLE 1 Cyclo[{4-(NH₂—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe]a) Synthesis of Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)—OH

L-hydroxyproline rnethylester hydrochloride is reacted with Fmoc-OSu inaqueous 1.0 N sodium carbonate/THF at room temperature. After completionof the reaction, Fmoc-Pro(4-OH)—OMe is isolated by precipitation.Fmoc-Pro(4-OH)—OMe is then added dropwise into a solution oftrisphosgene (0.6 eq.) in THF to give a chlorocarbonate intermediate.After 1 h dimethylaminopyridine (1.0 eq.) and N-Boc-diaminoethane (6.0eq.) are added and the reaction is stirred at room temperature. Aftercompletion of the reaction, the solvent is removed in vacuo and theresulting Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)—OMe is extracted from a twophase system of ethyl acetate/0.1 M HCl to give crude product (MH⁺=554)which is purified by crystallization from ethyl acetate. The methylester is then cleaved to the free acid by treatment with IN NaOH indioxane/water and the product Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)—OH ispurified on silica gel, [(M+Na)]⁺=562).

b) H-Phe-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-Phg-DTrp(Boc)-Lys(Boc)-Tyr(Bzl)—OH

Commercially available Fmoc-Tyr(Bzl)-O—CH₂—Ph(3-OCH₃)—O—CH₂-Polystyreneresin (SASRIN-resin, 2.4 mM) is used as starting material and carriedthrough a standard protocol consisting of repetitive cycles ofNa-deprotection (Piperidine/DMF, 2:8), repeated washings with DMF andcoupling (DIPCI: 4.8 mMIHOBT: 6mM, DMF). The following aminoacid-derivatives are sequentially coupled: Fmoc-Lys(Boc)-OH,Fmoc-DTrp(Boc)-OH, Fmoc-Phg-OH, Fmoc-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-OH,Fmoc-Phe-OH. Couplings (2 eq. amino acids) are continued or repeateduntil completion, i.e. until complete disappearance of residual aminogroups which is monitored by a negative ‘Kaiser’ Ninhydrin test. Beforecleavage of the completely assembled protected linear peptide from itsresin support the Na-Fmoc protection from the last residue is removed.

c) H-Phe-Pro(4-OCO—NH—CH₂—CH₂—NH-Boc)-Phg-DTrp(Boc)-Lys(Boc)-Tyr(Bzl)—OH

After washings with CH₂—Cl₂, the peptide-resin is transferred into acolumn or a stirred suction filter and the peptide fragment is cleavedand eluted with a short treatment with 2% TFA in CH₂Cl₂ within 1 h. Theeluate is immediately neutralized with a saturated NaHCO₃ solution. Theorganic solution is separated and evaporated and the side chainprotected precursor (MH⁺=1366) is cyclized without further purification.

d) cyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-Phg-DTrp-Lys-Tyr(Bzl )-Phe-],trifluoroacetate

The above linear fragment is dissolved in DMF (4 mM), cooled to minus 5°C. and treated with 2 eq. DIPEA then 1.5 eq. of DPPA and stirred untilcompletion (ca. 20 h) at 0-4° C. The solvent was almost completelyremoved in vacuo; the concentrate is diluted with ethyl acetate, washedwith NaHCO₃, water, dried and evaporated in vacuo.

For deprotection the residue is dissolved at 0° C. in TFA/H₂O 95:5 (ca.50 mM) and stirred in the mid for 30 min. The product is thenprecipitated with ether containing ca. 10 eq. HCl, filtered, washed withether and dried. In order to completely decompose remaining Indole-Ncarbaminic acid the product is dissolved in 5% AcOH and lyophilizedafter 15 h at ca. 5° C. Preparative RP-HPLC is earned out on a C-18 10μm STAGROMA column (5-25 cm) using a gradient of 0.5% TFA to 0.5% TFA in70% acetonitrile. Fractions containing the pure title compound arecombined, diluted with water and lyophilized. The lyophilisate isdissolved in water followed by precipitation with 10% Na₂CO₃ in water.The solid free base is filtered of, washed with water and dried invacuum at room temperature. The resulting white powder is directly usedfor the different salts.

EXAMPLE 2 Cyclo[{4-(NH₂—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe]in Salt Form

a. Acetate

Conversion to the acetate salt form is carried out using an ion-exchangeresin (e.g. AG 3-X4). MS (ESI): m/z 524.5 [M+2H]²⁺ [α]_(D) ²⁰=−42*,c=0.26 in AcOH 95%

b. Aspartate

Conversion to the mono- or di-aspartate is obtained by reacting 1equivalent of the compound of Example 1 with 1 or 2 equivalent ofaspartic acid in a mixture of acetonitrile/water 1:3. The resultingmixture is frozen and lyophilized. The di-aspartate may also be obtainedby dissolving the compound of Example 1 in water/acetonitrile 4:1,filtering, loading on a an ion-exchange resin, e.g. BioRad AG4X4 column,and eluting with water/acetonitrile 4:1. The eluate is concentrated,frozen and lyophilized. [α]_(D) ²⁰=−47.5°, c=2.5 mg/ml in methanol

c. Benzoate

Conversion to the benzoate may be obtained by dissolving the compound ofExample 1 with 2 equivalents of benzoic acid in a mixture ofacetonitrile/water 1:2. The resulting mixture is frozen and lyophilized.

d. Pamoate

1 equivalent of the compound of Example 1 is dissolved together with 1equivalent of embonic acid in a mixture of acetonitrile/THF/water 2:2:1.The resulting mixture is frozen and lyophilized.

EXAMPLE 3Cyclo[{4-(DOTA-NH—C₂H₄—NH—CO—O—)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)-Phe

a) cyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-Phg-DTrp-Lys(Cbo)-Tyr(Bzl)-Phe-],trifluoroacetate

The compound is synthesised in the same way likecyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-Phg-DTrp-Lys(Cbo)-Tyr(Bzl)-Phe-],trifluoroacetate by using Fmoc-Lys(Cbo)—OH instead of Fmoc-Lys(Boc)—OH.

b) 400 mg commercially available DOTA×2H₂O (SYMAFEX France) is dissolvedin 20 ml water. After addition of 20 ml DMF, 170 mgcyclo[-Pro(4-OCO—NH—CH₂—CH₂—NH₂)-Phg-DTrp-Lys(CBO)-Tyr(Bzl)-Phe-],together with 190 mg DCCI and 60 mg N-hydroxysuccinimide are added. Theresulting suspension is kept at room temperature for 72 hours. Afterfiltration, the solvent is removed under reduced pressure and theremaining crude is purified on silica gel (DCM/MeOH/HOAc_(50%)8/2/0.25-->7/3/1 as mobile phase).

c) For depratection the above DOTA conjugate is treated with 5 mltrifluoroacetic acid/thioanisole (9/1) for two hours at roomtemperature. After that the solution is poured into a mixture of 100 midiethylether+5 ml 3N HCl/diethylether and the resulting precipitate iasisolated by filtration. Purification is performed on silica gel usingDCM/Me0H/HOAc_(50%) 7/4/2-->7/514 as mobile phase. Analytically pureendproduct is obtained after a desalting step using a 0.1% TFA to 0.1%TEA in 90% CH₃CN gradient on a RP₁₈-HPLC column (Spherisorb 250×4.6 mm).MH⁺: 1434.7

Compound A in free form or in the form of pharmaceutically acceptablesalts and complexes exhibits valuable pharmacological properties asindicated in in vitro and in vivo tests and is therefore indicated fortherapy.

More particularly, Compound A exhibits an interesting binding profilefor human somatostatin receptors (hsst), particularly with respect tohsst1 , hsst2, hsst3 and hsst5. 5 somatostatin receptor subtypes, sst1,sst2, sst3, sst4 and sst5 have been cloned and characterized. hsst1,hsst2 and hsst3 and their sequences have been disclosed by Y. Yamada etal. in Proc. Nat. Acad. Sci., 89, 251-255 (1992). hsst4 and its sequencehave been disclosed by L. Rohrer et al. in Proc. Acad. Sol., 90,4196-4200 (1993). hsst5 and its sequence have been described by R.Panetta et al. in Mot. Pharmacol. 45, 417-427, 1993.

The binding assays may be carried out as disclosed hereunder usingmembranes from cell lines expressing selectively and stably hsst1,hsst2, hsst3, hsst4 or hsst5, e.g. CHO or COS cells.

Membranes are prepared according to known methods, e.g. as disclosed byC. Bruns et al. in Biochem. J., 1990, 65, page 39-44. Membranes preparedfrom hsst selective cell lines, e.g. CHO or COS cells stably expressinghsstl or hsst2 or hsst3 or hsst4 or hsst5 are incubated in triplicate ina total volume of 300 μl at 22° C. for 30 min with increasingconcentrations of [¹²⁵I-Tyr¹¹]-SRIF-14 in 10 mmol/l Hepes buffer (pH7.6) containing 0.5% BSA. The incubation is terminated by rapidfiltration and the filters are counted in a counter. Specific binding istotal binding minus non-specific binding in the presence of 1pmol/lsomatostatin-14. The experiments are carried out in triplicate.The affinity constant (K_(D)) and number of binding sites are calculatedusing appropriate statistics and graphical programs.

Compound A has in the above binding assays towards hsst1, hsst2, hsst3and/or hsst5 an IC₅₀ in the nMolar range, preferably an IC₅₀ of from 0.1to 10 nM (IC₅₀=concentration for half-maximal inhibition in acompetition binding assay using [¹²⁵I-Tyr¹¹]-SRIF-14 as hsst1-5 specificradioligand.

IC₅₀ hsst1 hsst2 hsst3 hsst4 hsst5 Compound 9.3 nM ± 1.0 nM ± 1.5 nM± >100 nM 0.16 nM ± A 0.1 0.1 0.3 0.1

Compound A also binds to growth hormone secretagogue receptors. Suchreceptors are disclosed by G. Muccioll et al., J. Endocrinol. 1998, 157,99-106, by H. Ong et al., in Endocrinology 1998, 139, 432-435 and byR.G. Smith et al., Horm. Res., 1999, 3), 1-8. The binding assay to thesereceptors may be carried out as disclosed in J. Endocrinol. Invest. 24:RC1-RC3, 2001. In this assay, Compound A displaces¹²⁵I-Tyr-Ala-hexarefin. Compound A is accordingly useful for modulatingthe activity of growth hormone secretagogue receptors, e.g. indicating apossible role in body weight gain or metabolic regulation.

Furthermore, Compound A shows GH-release inhibiting activity asindicated by the inhibition of GH release in vitro from culturedpituitary cells. For example, anterior pituitary glands from adult malerats are cut into small pieces and dispersed using 0.1% trypsin in 20 mMHEPES buffer. The dispersed cells are cultured for four days in MEM(Gibco) supplemented with 5% fetal calf serum, 5% horse serum, 1 mMNaHCO₃, 2.5 nM dexamethasone, 2.5 mg/m1 insulin and 20 U/mi Pen/Strep.On the day of the experiment the attached cells are washed two timeswith Krebs-Ringer medium buffered with 20 mM HEPES and supplemented with5 mM glucose and 0.2% BSA. Subsequently the cells are incubated forthree hours with Compound A in the presence of 3×10⁻¹⁰ M growth hormonereleasing factor. The amount of growth hormone released into the mediumis measured by RIA. Compound A has an IC₅₀ of 0.4 nM in this assay.

Compound A inhibits the release of growth hormone (GH) in rats. CompoundA is administered s.c. to anaesthetized rats. Blood is collected afterdecapitation 1 h after administration of the compound. The duration ofaction is estimated on the basis of the inhibition of basal GH secretion6 h after drug treatment. Hormone levels are measured by RlA 1 h and 6 hafter treatment. The ID₅₀-value for the inhibition of the hormonesecretion is determined graphically (log-probit) for each experiment andthe resulting values are averaged logarithmically. In this in vivo modelCompound A significantly inhibits growth hormone release with a longduration of action (Mean basal ID₅₀=5.5 μg/kg s.c. 6 h). In a similarassay for measuring the effect on insulin, Compound A inhibits insulinsecretion.

The potent and efficacious inhibition of GH was also confirmed in monkeystudies. Moreover, metabolic studies in diabetic monkeys demonstrated apotent antidiabetic/insulin-sensitizing effect of Compound A.

Furthermore, Compound A inhibits IGF-1 plasma levels in vivo asindicated in standard tests using male rats. Briefly, Compound A isadministered by osmotic pump implanted s.c. to male rats of a Lewisstrain. Blood samples are collected from the retrobulbar plexus using ashort anesthesia with e.g. isoflurane. In this assay, Compound Asignificantly lowers IGF-1 plasma levels with a long lasting effect:e.g. more than 60% inhibition is observed, after 14 days of treatmentwith 10 μg/kg/h of Compound A. More particularly no escape could beobserved after continuous treatment in rat recipients of aorta or kidneyallografts continuously infused with Compound A at 10 μg/kg/h up to 126days which induces a significant and persistent lowering of IGF-1 plasmalevels.

Compound A is accordingly useful for the prevention or treatment ofdisorders with an aetiology comprising or associated with excessGH-secretion and/or excess of IGF-1 e.g. in the treatment of acromegalyas well as in the treatment of type I or type II diabetes mellitus,especially complications thereof, e.g. angiopathy, diabeticproliferative retinopathy, diabetic macular edema, nephropathy,neuropathy and dawn phenomenon, and other metabolic disorders related toinsulin or glucagon release, e.g. obesity, e.g. morbid obesity orhypothalamic or hyperinsulinemic obesity. Compound A is also useful inthe treatment of enterocutaneous and pancreaticocutaneous fistula,irritable bowel syndrom, inflammatory diseases, e.g. Grave's Disease,inflammatory bowel disease, psoriasis or rheumatoid arthritis,polycystic kidney disease, dumping syndrom, watery diarrhea syndrom,AIDS-related diarrhea, chemotherapy-induced diarrhea, acute or chronicpancreatitis and gastrointestinal hormone secreting tumors (e.g. GEPtumors, for example vipomas, glucagonomas, insulinomas, carcinoids andthe like), lymphocyte malignancies, e.g. lymphomas or leukemias,hepatocellular carcinoma as well as gastrointestinal bleeding, e.gvariceal oesophagial bleeding.

Compound A is also useful in the treatment of tumors which aresomatostatin receptor positive, e.g. tumors bearing hsst1, hsst2, hsst3and/or hsst5, as indicated in proliferation tests with various cancercell lines bearing such somatostatin receptors.

The AR42J rat pancreatic tumor cell line is derived from anazaserine-induced exocrine pancreatic tumor (Jessop and Hay, 1980).Mycoplasma cell-free cultures are propagated in DMEM supplemented with10% fetal calf serum (FCS) at 5% CO₂. Cells are grown in the absence ofantibiotics or antifungal agents. Subconfluent AR42J cells aretrypsinized, diluted in DMEM+2.5% FCS and seeded in uncoated 96-wellplates. After a 48-hr incubation period (Day O), the number of cells ina separate control plate is determined both by counting cells in aCoulter counter and by the SRB colorimetric assay. The cells are thenexposed to Compound A for 2 to 5 days at various concentrations and thencounted. Under these conditions Compound A inhibits the proliferation ofthe tumor cells at concentrations ranging from 10⁻¹² to 10⁻⁶ M.

Tumor Growth Studies In Vivo

Female nude mice weighing 19-22 g are kept in groups of 5 animals andhave free access to drinking water and a pathogen-free rodent diet.Subcutaneous tumors are initiated from cultured AR42J cells. Treatmentcommences 2-4 days following inoculation of the tumor cells, Compound Ais administered as a continuous infusion, e.g. at a rate of 10 to 50μg/kg/hr. The size of the tumors is determined with a caliper. Forstatistical calculations Student's t-test is applied. In this assayCompound A inhibits tumor growth at day 11 by 51% vs saline control.

Compound A is thus useful for the treatment of malignant cellproliferative diseases, e.g. cancer tumors, particularly tumors bearingthe somatostatin receptor types to which it has a binding affinity, e.g.as disclosed hereunder for the complexed conjugated Compound A.

Compound A also has an inhibiting effect on angiogenesis, as indicatedin standard tests, e.g. in nude mice. Briefly, tumor cells (0.1 to10×10⁶ in 0.1 ml) (SiHa cells and MDA MB-231 cells prepared as disclosedin Angiogenesis, Ed. by R. Steiner, P. B. Weisz and R. Langer, 1992,Switzerland) are inoculated intracutaneously. Usually two midventralsites/mouse are injected which are located distant from the main ventralskin vessels so that the background vessel count is low. Control groupsreceive 0.1 ml 0.02 ID/c. trypan blue in PBS. 10 days followinginjection, anesthetized mice are sacrificed by CO₂ inhalation. The skinis mounted onto a plastic ring (40 mm diameter) for evaluation by aninverted microscope (Zeiss IM) at 12.5- and 25-fold magnification. As ameasure of angiogenesis, vessels are photographed and those are countedthat are directly connected with the tumor. In control animals thosevessels are counted that are connected to a defined area around theinjection site. This area corresponds to the mean area of the dermaltumors. The latter is determined by use of a caliper according to theequation 3.14×r². Compound A is administered s.c. either on the day oftumor inoculation or 3 days later. Control animals are vehicle-treated.In this assay, Compound A inhibits blood vessel formation whenadministered at a dose of e.g. 0.01 to 1000 μg/kg s.c.

Compound A is thus useful for the prevention or treatment ofangiogenesis, inflammatory disorders as indicated above includinginflammatory eye diseases , macular edema, e.g. cystoid macular edema,idiopathic cystoid macular edema, exudative age-related maculardegeneration, choroidal neovascularization related disorders andproliferative retinopathy.

Compound A also has an inhibiting effect on the proliferation andmigration of smooth muscle cells as indicated in following tests.

Chronic Allograft Rejection

The kidney of a male DA (RT1^(a)) rat is orthotopically transplantedinto a male Lewis (RT1¹) recipient. In total 24 animals aretransplanted. All animals are-treated with cyclosporine A at 7.5mg/kg/day per as for 14 days starting on the day of transplantation, toprevent acute cellular rejection. Contralateral nephrectomy is notperformed. Each experimental group treated with a distinct dose ofCompound A or placebo comprises six animals. Starting 14 days aftertransplantation, the recipient animals are treated up to 112 days byinfusion with Compound A or receive placebo. A_(t) 14 days aftertransplantation organ perfusion is measured by MRI. This is repeated atdays 53-64 after transplantation and at the end of the experiment. Theanimals are then autopsied. Administration of Compound A at a dose of 10μg/kg/h in this rat kidney allograft model results in an improved organperfusion as well as a reduction in chronic rejection related vascularremodelling and graft infiltration (cellular rejection). A marked andpersistent drop of IGF-1 levels has also been measured. These resultshave been confirmed in a second set of experiments using a heterotopicmouse heart allotransplantation model, demonstrating beneficial effectson vascular remodelling as well as in graft infiltration.

Compound A has also been tested in a carotid artery loop transplantationmodel using B10.A (2R) (H-2h²) mice as donor and 610.BR (H-2^(k)) miceas recipient. in brief, the donor carotid artery is transplantedparatopically as a loop into the recipient's carotid artery by anend-to-side anastomosis. A mini-pump is placed s.c. immediately aftertransplantation that delivers Compound A at a rate of 50 μg/kg/h.Carotid artery grafts are harvested at 30 days after transplantation toanalyse vascular remodeling e.g. by morphometric analysis of Verhoeffelastin stained paraffin sections using a computer-assisted system. Inthis model Compound A inhibits neointimal formation compared withnon-treated animals where a massive neointima is formed.

Angioplasty

Studies on angioplasty are done in the rat model of balloon catheterinjury. Balloon catheterization is performed on day 0, essentially asdescribed by Powell et al. (1989). Under Isofluorane anaesthesia, aFogarty 2F catheter is introduced into the left common carotid artery toobtain a uniform de-endothelialization. The catheter is then removed, aligature placed around the external carotid to prevent bleeding and theanimals allowed to recover. 2 groups of 12 RoRo rats (400 g,approximately 24 weeks old) are used for the study: one control groupand one group receiving Compound A and the rats are fully randomized.Compound A is administered by continuous infusion using minipumps at arate of 10 pg/kg/h starting 2 days before balloon injury (day-3) untilthe end of the study, 14 days after balloon injury. The rats are thenanaesthetized with Isofluorane and perfused with 0.1 M phosphatebuffered saline solution (PBS, pH 7.4) and then for 15 min. with 2.5%glutaraldehyde in phosphate buffer (pH 7.4). Carotid arteries are thenexcised, separated from surrounding tissue and immersed in 0.1 Mcacodylate buffer (pH 7.4) containing 7% saccharose and incubatedovernight at 4° C. The following day the carotids are then embedded inTechnovit 7100 according to the manufacturers recommendation. Thecross-sectional area of the media, neointima and the lumen are evaluatedmorphometrically by means of an image analysis system (MCID, Toronto,Canada). In this assay, Compound A inhibits neointimal thickeningsignificantly.

Compound A is thus also useful for preventing or combating graft vesseldiseases, e.g. alfa- or xenotransplant vasculopathies, e.g. graft vesselatherosclerosis, e.g. in a transplant of organ, e.g. heart, lung,combined heart-lung, liver, kidney or pancreatic transplants, or forpreventing or treating vein graft stenosis, restenosis and/or vascularocclusion following vascular injury, e.g. caused by catherizationprocedures or vascular scraping procedures such as percutaneoustransluminal angioplasty, laser treatment or other invasive procedureswhich disrupt the integrity of the vascular intima or endothelium.

Compound A has a beneficial plasma half-life. It has an eliminationhalf-life between 15 and 30 hours.

For all the above indications the required dosage will of course varydepending upon, for example, the host, the mode of administration andthe severity of the condition to be treated. in general, however,satisfactory results are obtained by administration in the order of from1 pg to 0.7 mg/kg/day of Compound A. An indicated daily dosage forpatients is in the range from about 2 pg to about 50 mg, preferablyabout 0.01 to about 40 mg, e.g. about 0.01 to about 3 mg s.c. of thecompound conveniently administered in divided doses up to 3 times a dayin unit dosage form containing for example from about 0.5 pg to about 25mg, e.g. from about 2 pg to 20 mg, for example from 2 pg to 1.5 mg ofthe Compound A.

Compound A may be administered in free form or in pharmaceuticallyacceptable salt form or complexes. Such salts and complexes may beprepared in conventional manner and exhibit the same order of activityas the free compound. The present invention also provides apharmaceutical composition comprising Compound A in free base form or inpharmaceutically acceptable salt form or complex form, together with oneor more pharmaceutically acceptable diluent or carrier. Suchcompositions may be formulated in conventional manner. Compound A mayalso be administered in sustained release form, e.g. in the form ofimplants, microcapsules, microspheres or nanospheres comprising e.g. abiodegradable polymer or copolymer, in the form of a liposomalformulation, or in the form of an autogel, e.g. a solid or semi-solidcomposition capable of forming a gel after interaction with patient'sbody fluids.

Compound A or a pharmaceutically acceptable salt or complex thereof maybe administered by any conventional route, for example parenterally e.g.in form of injectable solutions or suspensions (including e.g. thesustained release form as indicated above), orally using a conventionalabsorption enhancer, in a nasal or a suppository form or topically, e.g.in the form of an ophthalmic liquid, gel, oinment or suspensionpreparation, e.g a liposomal, microsphere or nanosphere formulation,e.g. for instillation or subconjunctival or intra- or peri-ocularinjections.

In accordance with the foregoing the present invention further provides:

1. Compound A or a pharmaceutically acceptable salt or complex thereoffor use as a pharmaceutical;2. A method of preventing or treating diseases or disorders as indicatedabove in a subject in need of such treatment, which method comprisesadministering to said subject an effective amount of Compound A or apharmaceutically acceptable salt or complex thereof; or3. Compound A or a pharmaceutically acceptable salt or complex thereoffor use in the preparation of a pharmaceutical composition for use inany method as defined under 2. above.

The conjugated Compound A or a pharmaceutically acceptable salt thereofis useful either as an imaging agent, e.g. visualisation of somatostatinreceptor positive tissues and cells e.g. somatostatin receptor positivetumors and metastases, inflammatory or autoimmune disorders exhibitingsomatostatin receptors, tuberculosis or organ rejection aftertransplantation, when complexed with a detectable element, e.g. a γ- orpositron-emitting nuclide, a fluorescent metal ion or a paramagneticion, e.g. ¹¹¹In, ¹⁶¹Tb, ¹⁷⁷Lu, ⁸⁵Y, ⁶⁸Ga Eu³⁺, Gd³⁺, Fe³⁺, Mn²⁺ or Cr²⁺,or as a radiopharmaceutical for the treatment in vivo of somatostatinreceptor positive tumors and metastases, rheumatoid arthritis and severeinflammation conditions when complexed with an α- or β-emitting nuclideor a nuclide with Auger-e-cascades, e.g. 90Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹¹At, ²¹³Bior ²⁰¹Tl, as indicated by standard tests.

In particular, it is observed that the conjugated Compound A binds tosomatostatin receptors with _(I)A values of from about 8 to 10. Compoundof Example 3 complexed with e g ¹¹¹In, ⁸⁸Y, ⁹⁰Y or ¹⁷⁷Lu binds in the nMrange to the respective sst sub-types in accordance with the bindingprofile of Compound A.

The affinity of the conjugated Compound A and its complexes forsomatostatin receptors can also be shown by in vivo testing, accordingto standard test methods, e.g. as disclosed in GB-A-2,225,579. Forexample the compound of Example 3 complexed with e.g. ¹¹¹In, ⁸⁸Y, ⁹⁰Y or¹⁷⁷Lu, gives a significant tumor accumulation 4 hours after injectioninto mice or rats bearing an exocrine pancreatic tumor expressing hsst2receptors.

After administration of a conjugated Compound A in complexed form, e.g.a ¹¹¹In, ¹⁷⁷Lu, ⁸⁶Y or ¹⁶¹Tb complexed Compound A, at a dosage of from 1to 5 μg/kg labelled with 0.1 to 5 mCi radionuclide, preferably 0.1 to 2mCi the tumor site becomes detectable.

The conjugated Compound A when radiolabelled with an α- or β-emittingradionuclide or a nuclide with Auger-e³¹ -cascades exhibits anantiproliferative and/or cytotoxic effect on tumor cells bearingsomatostatin receptors, e.g. as indicated in nude mice tests.

Nude mice are inoculated with AR42J rat pancreatic tumor cells orNCl—H69 human small cell lung cancer cells as disclosed above. Whentumors have reached a volume of 1 to 2 cm³ animals are randomized intocontrol and treatment groups. Conjugated Compound A in complexed form isadministered by i.p. or i.v. injections. Doses up to 40 mCi/kg are givenper mouse. The size of the tumors is determined with a caliper asdisclosed above. For statistical calculations Student's t-test isapplied. In this test, transient tumor shrinkage up to 50% of initial isobserved after one week and tumor growth is delayed for two weeks upon asingle application of the compound of Example 3 complexed with ⁹⁰Y. Incontrast the control groups showed continuous tumor growth with a volumedoubling time of about seven days.

Accordingly, in a series of specific or alternative embodiments, thepresent invention also provides:

4. Use of a conjugated Compound A complexed with a detectable elementfor in vivo detection of somatostatin receptor positive cells andtissues, e.g. somatostatin receptor positive tumors and metastasis, in asubject and recording the localisation of the receptors targeted by saidcomplex;5. A method for in vivo detection of somatostatin receptor positivetissues and cells, e.g. somatostatin receptor positive tumors andmetastasis, in a subject comprising administering to said subject aconjugated Compound A complexed with a detectable element, or apharmaceutically acceptable salt form, and recording the localization ofthe receptors targeted by said complex.

The conjugated Compound A in complexed form for use as an imaging agentmay be administered e.g. intravenously, e.g. in the form of injectablesolutions or suspensions, preferably in the form of a single injection.The radiolabelling may preferably be performed shortly beforeadministration to a subject.

In animals an indicated dosage range may be from 0.01 to 1 μg/kg of aconjugated Compound A complexed with 0.02 to 0.5 mCi γ-emittingradionuclide. In larger mammals, for example humans, an indicated dosagerange may be from 1 to 100 μg/m² conjugated Compound A complexed e.g.with 1 to 100 mCi/m²detectable element, e.g. ¹¹¹In, ⁸⁶Y or ¹⁷⁷Lu.

6. Use of a conjugated Compound A complexed with an a- or 8-emittingnuclide or a nuclide with Auger-&-cascades, for in vivo treatment ofsomatostatin receptor positive tumors and metastases.7. A method for in vivo treatment of somatostatin positive tumors andmetastases, e.g. for treating invasiveness of such tumors or symptomsassociated with such tumor growth, in a subject in need of suchtreatment which comprises administering to said subject atherapeutically effective amount of a conjugated Compound A complexedwith an a- or 8-emitting nuclide or a nuclide with Auger-e⁻ cascades.8. Use of a conjugated Compound A or a pharmaceutically acceptable saltthereof in the manufacture of an imaging agent or a radiopharmaceuticalcomposition.

Dosages employed in practising the radiotherapeutic use of the presentinvention will of course vary depending e.g. on the particular conditionto be treated, for example the known radiotoxicity to normal organsexpressing somatostatin receptors, the volume of the tumor and thetherapy desired. In general, the dose is calculated on the basis ofpharmacokinetik and radioactivity distribution data obtained in tohealthy organs and based on the observed target uptake. A β-emittingcomplex of a conjugated Compound A may be administered repeatedly e.g.over a period of 1 to 3 months.

In animals an indicated dosage range may be from 20 to 100 μg/kgconjugated Compound A complexed with 15 to 70 mCi of an α- or β-emittingnuclide or a nuclide with Auger-e⁻ cascades, e.g. ⁹⁰Y, ¹⁷⁷Lu or ¹⁶¹Tb.In larger mammals, for example humans, an indicated dosage range may befrom I to 100 μg/m² conjugated Compound A complexed e.g. with 1 to 100mCi/m² of an α- or β-emitting nuclide or a nuclide withAuger-e⁻-cascades, e.g. ⁹⁰Y, ¹⁷⁷Lu or ¹⁶¹Tb.

The conjugated Compound A in complexed form for use as aradiotherapeutic agent may be administered by any conventional route,e.g. intravenously, e.g. in the form of injectable solutions. It mayalso be administered advantageously by infusion, e.g. an infusion over15 to 60 min. Depending on the site of the tumor, it may be administeredas close as possible to the tumor site, e.g. by means of a catheter. Thepresent invention also provides a pharmaceutical composition comprisinga conjugated Compound A in free base form or in pharmaceuticallyacceptable salt form or complexed with a detectable or radiotherapeuticagent, together with one or more pharmaceutically acceptable diluent orcarrier.

Compound A or the conjugated Compound A in complexed form may besuitable for imaging or treating somatostatin receptor expressing oraccumulating tumors such as pituitary, gastro-enteropancreatic,carcinoids, central nervous system, breast, prostatic (includingadvanced hormone-refractory prostate cancer), ovarian or colonictumours, small cell lung cancer, malignant bowel obstruction,paragangliomas, kidney cancer, skin cancer, neuroblastomas,pheochromocytomas, medullary thyroid carcinomas, myelomas, lymphomas,Hodgkins and non-Hodgkins lymphomas, bone tumours and metastasesthereof, as well as autoimmune or inflammatory disorders, e.g.rheumatoid arthritis, Graves disease or other inflammatory eye diseases.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a conjugated Compound A or acomplex thereof together with one or more pharmaceutically acceptablecarriers or diluents therefor. Such compositions may be manufactured inconventional manner and may be presented, e.g. for imaging, in form of akit comprising two separate dosages, one being the radionuclide and theother the conjugated Compound A, with instructions for mixing them. Forradiotherapy, the conjugated Compound A in complexed form may preferablybe in the form of a hot liquid formulation.

Compound A or a conjugated Compound A in complexed form may beadministered as the sole active ingredient or in conjuction with, e.g.as an adjuvant to, other drugs. For example, Compound A may be used incombination with an immunosuppressive agent, e.g. a calcineurininhibitor, e.g. cyclosporin A or FK 506; a macrocyclic lactone havingimmunosuppressive properties, e.g. rapamycin or40-O-(2-hydroxyethyl)-rapamycin (RAD); an ascomycin havingimmunosuppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid or a salt thereof, e.g.Myfortic^(R); mycophenolate mofetil; 15-deoxyspergualine or animmunosuppressive homologue, analogue or derivative thereof; anaccelerating lymphocyte homing agent, e.g. FTY720; immunosuppressivemonoclonal antibodies, e.g., monoclonal antibodies to leukocytereceptors, e.g., MHC, CD2, CD3, C04, CD7, CD8, CD25, CD28, CD40, CD45,CD58, CD80, CD86 or to their ligands; other immunomodulatory compounds,e.g. a recombinant binding molecule having at least a portion of theextracellular domain of CTLA4 or a mutant thereof, e.g. an at leastextracellular portion of CTLA4 or a mutant thereof joined to a non-CTLA4protein sequence, e.g. CTLA4Ig (for ex. designated ATCC 68629) or amutant thereof, e.g. LEA29Y; adhesion molecule inhibitors, e.g. LFA-1antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4antagonists. Compound A may also be used in combination with ananti-inflammatory agent, a GH secretagogue receptor modulating agent,e.g. ghrelin or hexarelin, a GH receptor antagonist, e.g. pegvisomant,an insulin secretagogue or insulin secretion enhancer, e.g. a sulphonylurea, e.g. tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrolidinylarnino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-metanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide or tolylcyclamide, a short actingnonsuiphonyl urea, an oral insulinotropic agent derivative, e.g. a shortacting insulin enhancer, e.g. meglitinide, repaglinide, a phenyl aceticacid derivative, e.g.nateglinide, a DPP IV inhibitor, e.g.1-(2-[(5-cyanopyridin-2-yl)aminolethylamino)acetyl-(2S)-cyano-pyrrolidinedihydrochloride, LAF237, GLP-1 or a GLP-1 agonist analog, an insulinsenzitizer, e.g. a peroxisome proliferator activated receptor γ agonist(PPARγ), e.g. a glitazone, e.g.(S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione(englitazone),5-{[4-(3-(5-methyl-2-phenyl-4-oxazolyl)-1-oxopropyl)-phenyl]-methyl}-thiazolidine-2,4-dione(darglitazone),5-{[4-(1-methyl-cyclohexyl)methoxy)-phenyl]methyl}-thiazolidine-2,4-dione(ciglitazone),5-{[4-(2-(1-indolyl)ethoxy)phenyl]methyl}thiazolidine-2,4-dione(DRF2189),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy)]benzyl}-thiazolidine-2,4-dione(BM-13.1246), 5-(2-naphthylsulfonyl)-thiazolidine-2,4-dione (AY-31637),bis{4-[(2,4-dioxo-5-thiazolidinyl)-methyl]phenyl}methane (YM268),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl}-thiazolidine-2,4-dione(AD-5075),5-[4-(1-phenyl-1-cyclopropane-carbonylamino)-benzyl]-thiazolidine-2,4-dione(DN-108)5-{[4-(2-(2,3-dihydroindol-1-yl)ethoxy)phenyl]Methyl}-thiazolidine-2,4-dione,5-[3-(4-chloro-phenyl]-2-propynyl]-5-phenylsulfonyl)thiazolidine-2,4-dione,5-[3-(4-chlorophenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl)thiazolidine-2,4-dione,5-{[4-(2-(methyl-2-pyridinyl-amino)-ethoxy)phenyl]methyl)-thiazolidine-2,4-dione(rosiglitazone),5-{[4-(2-(5-ethyl-2-pyridyl)ethoxy)phenyl]-methyl}thiazolidine-2,4-dione(pioglitazone),5-{[4-((3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy)-phenyl]-methyl}-thiazolidine-2,4-dione(troglitazone),5-[6-(2-fluoro-benzyloxy)naphthalen-2-ylmethyl]-thiazolidine-2,4-dione(MCC555),5-{[2-(2-naphthyl)-benzoxazol-5-yl]-methyl}thiazolidine-2,4-dione(T-174) or5-(2,4-dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl-benzypbenzamide(KRP297), a non-glitazone type such as a N-(2-benzoylphenyl)-L-tyrosineanalogue, e.g. Gl-262570, or an oxolidinedione, e.g. JIT501, a dualPPARy/PPARa agonist, e.g. DRF-554158, NC-2100 or NN-622, a retinoid Xreceptor agonist or a rexinoid, e.g.2-[1-(3,5,5,8,8-pentarnethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid,4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid, 9-cis retinoic acid or an analog, derivative or a pharmaceuticallyacceptable salt thereof, a protein tyrosine phosphatase kinase 113, aglucogen synthase kinase-3 inhibitor, a non-peptidyl small moleculeinsulin mimetic compound, e.g. L-783,281 or CLX-901, or a low dose ofinsulin, a glutamine: fructose-6-phosphate amidotransferase inhibitor, aglucose-6-phosphatase inhibitor, a biguanide, e.g. Metformin, afructose-1,6-biphosphatase inhibitor, a glycogen phosphorylaseinhibitor, e.g. CP-91149, a glucagon receptor antagonist, e.g. CP-99711,NNC 92-1687, L-168,049 or BAY27-9955, a phosphoenolpyruvatecarboxykinase, a pyruvate dehydrogenase kinase inhibitor, anα-Glucosidase inhibitor, e.g.4″,6″-dideoxy-4″-[(1S)-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclo-hexenylamino}maltotrioseorO-4,6-dideoxy-4-{[1S,4R,5S,6S]-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]-amino}-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose(acarbose), N-(1,3-dihydroxy-2-propyl)valiolamine (voglibose) ormiglitol, or a gastric emptying inhibitor, e.g. GLP-1, CCK-8 and amylin(e.g. Pramlintide), an agent having anti-angiogenetic effects, e.g abenzoporphyrin, e.g. verteporfin, midostaurin or a4-pyridylmethyl-phtalazine.

Compound A or a conjugated Compound A in complexed form may also be usedin combination with an antiproliferative agent, e.g a chemotherapeuticdrug, e.g. paclitaxel, gemcitabine, cisplatinum, doxorubicin,5-fluorouracil or taxol, a hormonal agent or antagonist, e.g. ananti-androgen or mitoxantrone (especially in the case of prostatecancer), or an antiestrogen, like letrozole (especially in the case ofbreast cancer), an antimetabolite, a plant alkaloid, a biologicalresponse modifier, preferably a lymphokine or interferons, an inhibitorof protein tyrosine kinase and/or serine/threonine kinase, or an agentwith other or unknown mechanism of action, e.g.any epothilone orepothilone derivative, or a macrocyclic lactone, e.g. rapamycin, RAD orCC1779.

Where Compound A or a conjugated Compound A in complexed form isadministered in conjunction with another drug, dosages of theco-administered drug will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the condition to betreated, and so forth. The terms “co-administration” or “combinedadministration” or the like as utilized herein are meant to encompassadministration of the selected therapeutic agents to a single patient,and are intended to include treatment regimens in which the agents arenot necessarily administered by the same route of administration or atthe same time.

In accordance with the foregoing the present invention provides in a yetfurther aspect:

9. A pharmaceutical combination comprising a) a first agent which isCompound A or a conjugated Compound A in complexed form and b) aco-agent, e.g. as defined above.10. A method as defined above comprising co-administration, e.g.concomitantly or in sequence, of a therapeutically effective amount ofCompound A or a conjugated Compound A in complexed form, and a seconddrug substance, said second drug substance being, e.g. as indicatedabove.

The particular combination of the invention will be selected dependingon the prevention or treaternent of diseases or disorders; e.g. acombination with an immunosuppressive agent for e.g. the prevention ortreatement of chronic graft rejection, a combination with an insulinsecretagogue, insulin secretion enhancer, insulin sensitizer or a lowdose of insulin in the treatment of diabetes or complications thereof, acombination with an anti-inflammatory agent for the prevention ortreatment of inflammatory diseases or disorders, a combination with anagent having anti-angiogenetic effects for the prevention or treatmentof e.g. macular edema or degeneration or in cancer, a combination with achemotherapeutic agent for use in cancer.

1. A method of treating somatostatin receptor expressing or accumulatingtumors such as pituitary, gastro-enteropancreatic, central nervoussystem, breast, prostatic (including advanced hormone-refractoryprostate cancer), ovarian or colonic tumours, small cell lung cancer,malignant bowel obstruction, paragangliomas, kidney cancer, skin cancer,neuroblastomas, pheochromocytomas, medullary thyroid carcinomas,myelomas, lymphomas, Hodgkins and non-Hodgkins lymphomas, bone tumoursand metastases thereof, as well as autoimmune or inflammatory disorders,e.g. rheumatoid arthritis, Graves disease or other inflammatory eyediseases in a subject, wherein said method comprises administering aneffective amount of a compound of formula

where one of the amino groups being optionally in protected form, or asalt or a complex thereof.
 2. The method according to claim 1 whereinsaid somatostatin receptor expressing or accumulating tumor is amalignant bowel obstruction.
 3. The method according to claim 1 whereinsaid somatostatin receptor expressing or accumulating tumor is aneuroendocrine tumor.
 4. A method of treating gastrointestinal hormonesecreting tumors in a subject, wherein said method comprisesadministering an effective amount of a compound of formula

where one of the amino groups being optionally in protected form, or asalt or a complex thereof.
 5. The method according to claim 4 whereinsaid gastrointestinal hormone secreting tumor is a carcinoid tumor.